Summary

Neurons acquire distinct cell identities and implement differential gene
programs to generate their appropriate neuronal attributes. On the basis of
position, axonal structure and synaptic connectivity, the 302 neurons of the
nematode Ceanorhabditis elegans are divided into 118 classes. The
development and differentiation of many neurons require the gene
zag-1, which encodes a δEF1/ZFH-1 Zn-finger-homeodomain
protein. zag-1 mutations cause misexpression of neuron-specific
genes, block formation of stereotypic axon branches, perturb neuronal
migrations, and induce various axon-guidance, fasciculation and branching
errors. A zag-1-GFP translational reporter is expressed transiently
in most or all neurons during embryogenesis and in select neurons during the
first larval stage. Analysis of the zag-1 promoter reveals that
zag-1 is expressed in neurons and specific muscles, and that ZAG-1
directly represses its own expression. zag-1 activity also
downregulates expression of genes involved in either the synthesis or reuptake
of serotonin, dopamine and GABA. We propose that ZAG-1 acts as a
transcriptional repressor to regulate multiple, discrete, neuron-specific
aspects of terminal differentiation, including cell migration, axonal
development and gene expression.

INTRODUCTION

Cell signaling and cell-intrinsic processes orchestrate the specification
and differentiation of distinct neuron types
(Edlund and Jessell, 1999).
For example, a gradient of sonic hedgehog establishes the expression pattern
of different transcriptional regulators along the dorso-ventral axis in the
developing, vertebrate ventral spinal cord. The unique complement of factors
in each domain determines, in part, the subsequent fate adopted by each
neuron. Presumably, it also initiates transcriptional cascades that generate
the appropriate characteristics for an individual neuron type, such as axonal
structure, synaptic connections and differentiated gene-expression profile.
Although factors, such as LIM-homeodomain and ETS-domain proteins, which
contribute to the establishment of axonal-projection patterns and
connectivity, have been identified in several organisms (reviewed by
Hobert and Westphal, 2000;
Shirasaki and Pfaff, 2002),
the repertoire of factors needed to generate attributes for most neuron types
is largely unknown.

The nervous system of C. elegans consists of relatively few
neurons but it contains a great diversity of neuron types. The 302 neurons of
the adult hermaphrodite are grouped classically into 118 classes, based on
position, axonal morphology and connectivity
(White et al., 1986).
Subsequent studies indicate that individual neurons in some classes exhibit
different gene-expression profiles, revealing the potential for a still
greater number of distinct neuron types
(Troemel et al., 1999;
Yu et al., 1997). Several
genes controlling the formation of neuron-specific characteristics and
functional properties have been discovered. Some genes specify the fate of a
neuron from an alternative or default state: mutation of the LIM-homeodomain
gene lim-4 causes AWB to adopt an AWC-like fate
(Sagasti et al., 1999); and
mutation of the forkhead domain gene unc-130 induces ASG to adopt an
AWA-like fate (Sarafi-Reinach and
Sengupta, 2000). Other genes define the characteristics of
different neuron types: the homeodomain gene unc-42 regulates axonal
development and neuron-type-specific expression of several glutamate and
chemosensory receptors (Baran et al.,
1999; Brockie et al.,
2001); the homeodomain gene unc-30 controls
differentiation of GABAergic D-type motor neurons and unc-30 mutants
have defects in axon pathfinding, synaptic connectivity and expression of the
glutamic acid decarboxylase UNC-25 and GABA vesicular transporter UNC-47
(Eastman et al., 1999;
Jin et al., 1994).

δEF1/ZFH-1 Zn-finger-homeodomain proteins are distinguished by two
arrays of highly similar C2H2-type Zn-finger domains and a centrally located
homeodomain, and can act as transcriptional repressors through recruitment of
the corepressor C-terminal binding protein (CtBP). Although
Drosophila has one δEF1/ZFH-1 homolog
(Fortini et al., 1991),
vertebrates have two, one of which is most similar to the eponymous chickδ
EF1 (Funahashi et al.,
1993) (also known as Nil-2-a, TCF8, ZEB, BZP, AREB6, MEB1 and
ZFHEP) (Sekido et al., 1996),
the second, SIP1 (also known as ZEB-2), can associate with Smad proteins
(Postigo and Dean, 2000;
Verschueren et al., 1999).
Drosophila zfh-1 is expressed in embryonic mesoderm, mesodermally
derived tissues and motor neurons (Lai et
al., 1991), and is needed for development of gonadal mesoderm,
heart and other tissues derived from mesoderm
(Broihier et al., 1998;
Lai et al., 1993;
Su et al., 1999). Mouseδ
EF1 is expressed in notochord, somites, limb, neural-crest derivatives
and some CNS regions, and is required for thymus and skeletal development
(Higashi et al., 1997;
Takagi et al., 1998). Althoughδ
EF1 is a negative regulator of muscle differentiation in vitro
(Postigo and Dean, 1997),
mouse δEF1 knockouts lack muscle and CNS defects. Mouse SIP1 is needed
for development and migration of specific neural-crest cells
(Van De Putte et al.,
2003).

Here we report the genetic and molecular characterization of the lone
C. elegans δEF1/ZFH-1 homolog, ZAG-1. zag-1 is
essential for many aspects of neuronal differentiation and is expressed widely
and dynamically during formation of the nervous system and in some muscles.
ZAG-1 represses its own expression by interacting with conserved sequences in
its promoter and, possibly, introns. We propose that zag-1 acts as a
transcriptional repressor during the late stages of neuronal differentiation
to establish several neuron-specific characteristics, including correct cell
position and axon structure, and proper expression of cell-surface proteins,
transmembrane receptors, ion channels, and biosynthetic enzymes and reuptake
transporters for neurotransmitters.

We recovered pag-3 and zag-1 alleles in a screen for
mutants with defects in PVQ, which will be described in detail elsewhere. In
brief, sra-6::gfp(oyIs14) parental generation (P0) animals were
treated with either ethyl methanesulfonate (EMS) or N-ethyl-N-nitrosourea
(ENU) (De Stasio et al.,
1997), F2 progeny that exhibited behavioral defects, such as an
uncoordinated movement (Unc) or egg-laying defective (Egl) phenotype, were
transferred individually to new plates and their progeny examined using
epifluorescence microscopy to identify mutants with PVQ cell-fate
specification, axonal development and other defects. We picked Uncs and Egls
to enrich for animals with potential abnormalities in the structure or
function of the nervous system.

Five pag-3(zd48, zd49, zd63, zd111 and zd120)
and two zag-1(zd85 and zd86) alleles were recovered
following EMS treatment and one pag-3(zd124) allele was found
following ENU treatment. All six pag-3 mutations mapped to
LGX and failed to complement pag-3(ls20) for the Unc
phenotype. Two-factor crosses of zd85 and zd86 indicated
linkage to lin-1 IV. Complementation testing indicated that
zd85 and zd86 are allelic and defined a new gene,
zag-1. We performed three-factor crosses to map zag-1
further. From zag-1(zd85)/lin-1 dpy-13 heterozygotes, 24 out of 27
Lin non-Dpy recombinants segregated Zag-1 animals and from
zag-1(zd86)/lin-1 dpy-13 heterozygotes, 20 out of 21 Lin non-Dpy
recombinants segregated Zag-1 animals. From vab-2/lin-1 zag-1(zd85)
heterozygotes, 2 out of 40 Zag non-Lin recombinants segregated Vab-2 animals
and 30 out of 35 Lin non-Zag recombinants segregated Vab-2 animals. Thus,
zag-1 maps between vab-2 and dpy-13.

zag-1 rescue

Germline transformation (Mello and
Fire, 1995) of zag-1 mutants was performed by
co-injecting test DNA (10-50 μg ml-1) and
sur-5::SUR-5-GFP reporter DNA (50 μg ml-1), pTG96,
which labels most nuclei with GFP (Yochem
et al., 1998). Transformed F1 and F2 animals were identified by
GFP expression from pTG96. The cosmids F28F9, F47C12, W02C12, W03D2, F49F1,
R07C12 and R08D10 were tested, either individually or in pools, for rescue of
the Unc phenotype of zag-1(zd85) animals. Three out of five
extrachromosomal array lines containing F28F9 and none of the lines harboring
the other cosmids were rescued. Subclones of F28F9 were constructed using
pBluescript II KS(-). The 10 kb KpnI-SalI genomic fragment
(pSK55; position 3,851,820-3,861,821 of chromosome IV) rescued (3/3
lines), whereas an 8.8 kb PstI-PstI fragment (pSK54;
position 3,852,925-3,861,764) failed to rescue (0/2 lines).

GFP transgenics

Transcriptional and translational reporter lines were generated using
GFP-expression vectors provided by A. Fire. DNA sequences upstream of each
gene were amplified from N2 genomic DNA using PCR, and cloned in-frame into an
appropriate GFP vector. For dat-1::gfp, DNA sequences -3671 to +1
were amplified (+1 is first base of ATG) and cloned into SphI and
MscI sites of pPD95.77. For talin::gfp, DNA sequences -2070
to +6 were amplified and cloned into SphI and BamHI sites of
pPD95.77. For tph-1::gfp, DNA sequences -1748 to +9 were amplified
and cloned into HindIII and MscI sites of pPD95.81. For
unc-129::gfp, DNA sequences -3087 to +6 were amplified and cloned
into XbaI and MscI sites of pPD95.75. Sequences were
included in downstream primers to generate a BamHI, MscI or
StuI (unc-129::gfp) site. glr-1::gfp, mec-4::gfp
and odr-2::cfp were derived from previously described reporters
(Chou et al., 2001;
Lai et al., 1996;
Maricq et al., 1995) by
replacement of `wild type' GFP sequences with GFP S65C or CFP (GFP Y66W,
N146I, M153T and V163A) coding sequences with synthetic introns.

Using PCR and standard cloning methods, we introduced an XbaI site
at the end of exon 7 (TCTACCTAG changed to TCTAGATAG)
and fused GFP gene and unc-54 3′ untranslated region (UTR)
sequences from pPD95.77 to zag-1 genomic sequences.
zag-1::ZAG-1-GFP is predicted to produce a full-length ZAG-1-GFP
fusion protein that includes a substitution of arginine for the C-terminal
threonine. We made zag-1::ZAG-1(213)-GFP, which is expected to
generate a ZAG-1(213)-GFP fusion protein containing the N-terminal 213 amino
acids of ZAG-1, by cloning the GFP gene and unc-54 3′UTR
sequences from pPD95.77 into the BamHI site in exon 2.

Images were acquired using a Hamamatsu Orca CCD camera and a Leica DMRE
microscope, and edited using Improvision Openlab and Adobe Photoshop.

Sequence analysis

We determined the DNA sequence of the longest predicted EST clone, yk312a9,
of F28F9.1. DNA sequences were determined using the ABI PRISM BigDye
Terminators Cycle Sequencing kit and either an ABI PRISM 310 Genetic Analyzer
or MJ Research BaseStation 51 DNA Fragment Analyzer. The genomic organization
of F28F9.1 based on the sequence of yk312a9 is different from the hypothetical
gene structure in WormBase release WS99 (17 April, 2003)
(http://wormbase.org);
we believe the yk312a9-based structure is correct because it is derived from
cDNA sequence and the open reading frames are highly conserved in the related
nematode C. briggsae.

To find alterations associated with zag-1 mutations, the coding
and splice-junction regions were amplified from N2, zd85 and
zd86 genomic DNA using PCR and flanking primers. The DNA sequences of
the amplified products were determined directly using either internal
sequencing primers or the PCR primers and a cycle-sequencing protocol.
zd85 and zd86 each contained a single point mutation within
the F28F9.1 gene and the N2 F28F9.1 sequence was identical to that reported by
the C. elegans Sequencing Consortium.

RESULTS

zag-1 identification

We discovered zag-1 in a screen for mutations that disrupt PVQ
development. PVQ is a pair of interneurons located in the lumbar ganglia in
the tail, and each extends a single axon that runs within the ventral nerve
cord (VNC) to the nerve ring in the head
(White et al., 1986). An
sra-6::gfp (serpentine receptor class A) reporter is expressed in PVQ
and in amphid neurons ASH and ASI (Troemel
et al., 1995), which allows direct observation of these neurons in
living animals using epifluorescence microscopy
(Fig. 1A). We treated
sra-6::gfp(oyIs14) animals with the mutagens EMS and ENU and
identified mutants with PVQ defects (see Materials and Methods). Expression of
sra-6::gfp was seen uniformly in ASH and ASI but was observed rarely
in both PVQs in eight mutants (Fig.
1B, Table 1, data
not shown). Six mutations (zd48, zd49, zd63, zd111, zd120 and
zd124) are pag-3 alleles. We reported previously that PVQ is
present in pag-3 mutants but fails to express sra-6::gfp
(Cameron et al., 2002).

Effects of different concentrations of ASIP morpholinos on mitotic
orientation in neural tube stage

The remaining two alleles (zd85 and zd86) defined a new
gene, zag-1 IV. Both are recessive and exhibit a similar,
uncoordinated, behavioral phenotype. zag-1 worms were active but,
typically, had a kinked appearance and serious difficulty moving backward. The
penetrance and expressivity of the PVQ sra-6::gfp expression defect
were incomplete (Table 1). PVQ
axons, when detected, followed their normal trajectory and 67 out of 68
reached the nerve ring. We examined zag-1 animals using DIC
microscopy and observed neuronal nuclei in positions characteristic of PVQ,
which indicated that the lack of GFP-labeled neurons resulted from either
reduction or elimination of sra-6::gfp expression rather than the
absence of PVQ.

lin-11::gfp(mgIs21) has faint PVQ expression in young
larvae (Hobert et al., 1998),
permitting visualization of cell bodies but not axons. We detected both PVQ
cell bodies in zag-1; lin-11::gfp larvae (21/21 for mgIs21,
19/19 for zd85; mgIs21 and 23/23 for zd86; mgIs21),
confirming the presence of PVQ and revealing the proper regulation of
lin-11::gfp expression. Together, these observations indicate that
zag-1 is needed to specify some features of PVQ-cell fate, such as
the expression of sra-6, but not lin-11.

HSN differentiation requires zag-1

The HSN serotoninergic motor neurons are born in the tail and migrate to
flank the gonad in the midbody during embryogenesis
(Desai et al., 1988;
Sulston et al., 1983). HSN
matures during the fourth larval (L4) stage: the nucleus and nucleolus enlarge
and a distinctive structure, called the `hood', forms around the nucleus. The
HSN axons go round the vulva, enter the VNC and continue to the nerve
ring.

Serotoninergic neurons express the tryptophan hydroxylase TPH-1
(Sze et al., 2000).
tph-1::gfp(zdIs13) expressed GFP in serotoninergic neurons ADF, HSN,
NSM and male-specific CP and R(1,3,9) (Fig.
1C). GFP expression in HSN was observed first during late L4 and
continued throughout adulthood, consistent with the immunological detection of
serotonin that starts in young adults
(Desai et al., 1988). We found
that HSN GFP expression was rare or absent in zag-1 tph-1::gfp adult
animals, whereas expression was unchanged in ADF, NSM, CP and R(1,3,9)
(Fig. 1D,
Table 1).
unc-86::gfp(kyIs179) displays GFP expression in HSN during larval
development (Gitai et al.,
2003). We examined zag-1(zd85); unc-86::gfp
animals and observed both HSNs in their proper midbody location (20/20 for
kyIs179 and 18/18 for zd85; kyIs179). Thus, the HSNs are
born, migrate to the midbody and express unc-86 but not
tph-1 in zag-1 mutants.

odr-2 encodes a GPI-linked, cell-surface protein that is expressed
by several sensory neurons, interneurons and motor neurons but not HSN
(Chou et al., 2001).
CFP-expressing HSNs were never observed in wild-type odr-2::cfp
adults (n=50), whereas CFP-expressing HSNs were detected in all
zag-1; odr-2::cfp adults (zd85; 34/40, 2 HSNs, 6/40, 1 HSN
and zd86; 38/40, 2 HSNs, 2/40, 1 HSN). The morphology of HSN cell
bodies and axons was typically abnormal in zag-1 mutants visualized
using either odr-2::gfp or unc-86::gfp. The HSNs retained a
generic neuronal appearance and, often, failed to form their characteristic
hood. Although most HSN axons extended ventrally and entered the ventral
nerve, their trajectory was aberrant and they often branched extensively;
ectopic axons also projected from cell body
(Fig. 1E-G). In summary,
zag-1 mutants have defects in multiple aspects of HSN
differentiation, including axon pathfinding, hood formation and tph-1
expression. HSN development appears wild type until midlarval stages: the HSNs
are born, migrate to the midbody and correctly express unc-86 and not
odr-2.

zag-1 mutants lack specific axon branches

Although the axons of most neurons in C. elegans are relatively
simple and unbranched, the axons of a few neurons have well-defined,
reproducible, branched structures (White
et al., 1986). The dopaminergic neurons ADE and PDE have axons
with stereotypic branched structures and can be visualized using a GFP
reporter to the dopamine transporter gene dat-1
(Nass et al., 2001). ADE is a
pair of ciliated neurons located behind the second bulb of the pharynx. Each
ADE projects a process ventrally that extends to the ventral ganglion and a
process anteriorly that splits to a form a ciliated ending and a branch that
enters the ring neuropil laterally (Fig.
2A,I). We found that the anterior process always formed a branch
to the ring neuropil in wild-type dat-1::gfp(zdIs31) animals (47/47
ADEs) but this was generated infrequently in zag-1; dat-1::gfp
animals (8/100 ADEs zd85; zdIs31, 17/100 ADEs zd86, zdIs31)
(Fig. 2B,I). The ciliated
branch of the anterior process and the ventral process were unaffected,
indicating that zag-1 mutants have a specific defect in branch
formation rather than a general defect in ADE-axon outgrowth.

PDE is a pair of ciliated neurons located in the posterior body. Each PDE
projects a process dorsally with a ciliated ending, and a process ventrally
that enters the VNC, splits and extends anteriorly and posteriorly
(Fig. 2C,I). Because the PDEL
and PDER axons run together in the right ventral nerve bundle, we scored the
extent of growth of the longest process. The posterior branch of the ventral
PDE process typically extended ∼3/4 of the distance between PDE cell body
and anus in wild-type dat-1::gfp animals (38/47 full length), but
occasionally it was shorter (9/47 partial length) or failed to extend (2/47).
By contrast, most zag-1; dat-1::gfp animals lacked the posterior
branch (zd85; 3/100 full-length, 35/100 partial length, 62/100 no
extension, and zd86; 1/100 full length, 26/100 partial length, 73/100
no extension) (Fig. 2D,I). The
posterior branch was rarely redirected anteriorly. The anterior branch was
slightly shorter than wild type in ∼1/2 of the zag-1; dat-1::gfp
animals and the ciliated dorsal process was unchanged. We conclude that
zag-1 is required for the extension of the anteriorly directed axon
branches of ADE and the posteriorly directed axon branches of PDE.

The HSN axons often form one or more short branches near the vulva
(White et al., 1986). The HSN
axons detected in zag-1(zd85) tph-1::gfp animals failed to branch at
the vulva (0/17 HSNs) and were sometimes short or slightly misdirected (5/17),
whereas most HSNs in wild-type tph-1::gfp animals formed a branch
near the vulva (25/31) and were rarely short or misdirected (2/200). Most HSN
axons in zag-1 mutants had severe guidance defects viewed using
either odr-2::cfp or unc-86::gfp (see above). The few
GFP-expressing HSNs detected in zag-1(zd85) tph-1::gfp animals
presumably represented a population with sufficient zag-1 activity to
promote tph-1::gfp expression and the formation of a nearly correct
axon in most cases. However, the complete absence of stereotypic branches at
the vulva argues that zag-1 is needed to shape multiple, discrete
HSN-axon features, such as the extension of an axon with an appropriate nerve
ring trajectory and formation of branches at the vulva.

The six DD and 13 VD motor neurons are located in the VNC. Each extends a
process anteriorly that forms a collateral to the dorsal nerve cord and
divides and projects both anteriorly and posteriorly (DD also extends a short
process posteriorly) (Fig. 2I).
DD1 and VD2 commissures extend on the left side and, typically, fasciculate,
whereas DD2-6 and VD1,3-13 commissures extend on the right side. Many DD and
VD axons terminated their growth prematurely, were misdirected and branched
abnormally in zag-1 mutants; the anteriorly directed branches
adjacent to dorsoventral commissures were also often missing
(Fig. 2I, data not shown). In
addition, many DD and VD commissures extended on the wrong side. DD1 and VD2
commissures were fasciculated and extended on the left side in wild-type
unc-25::gfp(juIs75) animals (n=63) and, infrequently, a
single, additional commissure extended inappropriately on the left side
(12/63). By contrast, 74% of zag-1(zd85); unc-25::gfp animals had one
or more inappropriate commissures on the left side (19/50 one, 11/50 two, 6/50
three and 1/50 four). Conversely, DD1 and VD2 often extended on the right side
(5/50 both, 35/50 one). Thus, zag-1 is needed for the formation of
anteriorly directed DD and VD axon branches as well as for several aspects of
DD and VD axon guidance.

Other zag-1 axon outgrowth defects

Several interneurons and motor neurons, including the command interneurons
AVA, AVB, AVD, AVE and PVC that extend processes along the VNC, express the
GLR-1 glutamate receptor (Hart et al.,
1995; Maricq et al.,
1995), whereas a subset of glr-1::gfp-expressing neurons,
AVA, AVD, AVE, AVG, PVC and RIM, express the NMR-1 glutamate receptor
(Brockie et al., 2001). We
found that all zag-1 glr-1::gfp(zdIs3) animals (40/40 zd86)
had three or more aberrant axons emanating from either the nerve ring or tail
that zigzagged and branched profusely along the length of the animal, but 1
out of 30 wild-type glr-1::gfp animals had a single, short,
inappropriate lateral process that exited the nerve ring
(Fig. 2E,F). Ectopic, neuronal
expression was detected in the tail and the nerve ring was disorganized and
defasciculated. The VNC was also defasciculated in zag-1; nmr-1::gfp
animals, but the growth of ectopic axons in lateral positions was uncommon.
Thus, most of the aberrant processes seen in zag-1 glr-1::gfp animals
likely arose from glr-1::gfp-expressing neurons that do not express
nmr-1::gfp. zag-1 mutations also caused variable expression of
nmr-1::gfp in PVC and ectopic expression in an unpaired tail neuron.
These results indicate that zag-1 is required for specification and
axon guidance of interneurons and motor neurons that express glr-1
and nmr-1, and to prevent misexpression of glr-1 and
nmr-1.

AVG, an interneuron located in the retrovesicular ganglion, pioneers the
right ventral nerve bundle and is thought to provide cues that promote the
proper assembly and organization of VNC (R. M. Durbin, PhD thesis, University
of Cambridge, UK, 1987). Ablation of the parent of AVG led to inappropriate
growth of DD and VD commissures on the left side as well as extension of many
longitudinal axons on the wrong side of the VNC. We found that AVG sometimes
terminated its growth prematurely in zag-1; odr-2::cfp animals (14/40
zd86), but it extended completely in wild-type odr-2::cfp
animals. As such, the disorganization of VNC and extension of DD and VD
commissures on the wrong side might result in part from defects in AVG
outgrowth.

ALM migration and axonal development require zag-1

The six touch receptor neurons, ALM, PLM, AVM and PVM, are needed for the
gentle touch response (Chalfie et al.,
1985) and express the MEC-4 ion channel subunit
(Lai et al., 1996). The two
ALMs are born and migrate to characteristic anterior body positions during
embryogenesis (Sulston et al.,
1983). In wild-type mec-4::gfp(zdIs5) animals, 49 out of
50 ALM cell bodies were found ∼3/4 of the way between the second bulb of
pharynx and the vulva in young adults and 1 out of 50 was found halfway or
less. By contrast, 12 out of 49 ALMs in zdIs5; zd85 and 11 out of 45
ALMs in zdIs5; zd86 animals were positioned halfway or less, and many
had a variable dorsoventral position, indicating that zag-1 is needed
for ALM to complete its posterior migration. Occasionally, mec-4::gfp
expression in ALM was not seen in zag-1 mutants (50/50
zdIs5, 49/50 zdIs5; zd85 and 45/50 zdIs5;
zd86).

Each ALM projects a short axon posteriorly and an axon anteriorly that runs
at least to the first bulb of pharynx and forms a branch that enters neuropil
and contacts the AVM branch. We found that all anterior ALM axons (50/50) had
a wild-type trajectory in mec-4::gfp(zdIs5), whereas only 9 out of 50
were wild type in zdIs5; zd86 animals
(Fig. 2G-I). Most anterior ALM
axons (41/50) grew as far as the nerve ring and turned to join the neuropil.
The nerve ring branch appeared tangled and often failed to contact the AVM
branch, which was also disorganized. Anterior ALM processes in zdIs5;
zd85 animals were similarly defective. The posterior ALM process was
typically absent in wild-type mec-4::gfp and mec-4::gfp;
zag-1 animals, which likely represents natural variation. These
observations indicate that zag-1 is required to form the ALM-axon
extension past the nerve ring. Thus, zag-1 is needed for multiple
phases of ALM development, including cell migration, axonal development and
mec-4 expression. mec-4::gfp expression and axon morphology
of PLM, the posterior homolog of ALM, were essentially wild type in
zag-1 mutants.

AVM and PVM are descendants of neuroblasts QR and QL, respectively. QR and
its descendants migrate to specific positions in the anterior body, whereas QL
and its descendants migrate to stereotypic positions in the posterior body.
AVM and PVM were found in their correct locations in zag-1 animals
but mec-4::gfp expression was altered. PVM mec-4::gfp
expression was often eliminated (25/25 zdIs5, 15/25 zdIs5;
zd85 and 10/25 zdIs5; zd86) but AVM mec-4::gfp
expression was largely unaffected (25/25 zdIs5, 24/25 zdIs5;
zd85 and 24/25 zdIs5; zd86). Inappropriate expression of
odr-2::cfp was also observed occasionally in PVM (data not shown).
AVM and PVM axons enter the VNC and run anteriorly, AVM continues past the
first bulb of pharynx and PVM stops in the anterior body. AVM forms a branch
that splits, enters the neuropil and contacts ALM branches. Only 8 out of 24
AVM processes extended past the first bulb of pharynx in mec-4::gfp;
zag-1(zd86) compared to 25 out of 25 in wild-type mec-4::gfp
(Fig. 2G-I). Sixteen out of 24
AVM axons turned to enter the nerve ring after passing the second bulb of
pharynx and lacked an anteriorly directed lateral extension, similar to the
phenotype displayed by ALM axons. AVM branches were disorganized and often
failed to make contact with ALM processes. PVM axons appeared wild type.
Therefore, zag-1 mutations had differential effects on AVM and PVM.
AVM usually expressed mec-4 and had distinct axon-branch-formation
defects, whereas PVM often failed to express mec-4 and had a
wild-type axon structure when detected.

unc-25 encodes the GABA biosynthetic enzyme glutamic acid
decarboxylase and is expressed in all 26 GABAergic neurons
(Jin et al., 1999). The
unc-25::gfp(juIs75) reporter, which has 1.8 kb of sequence 5′
of the ATG and coding sequences for the first 13 amino acids of UNC-25,
expresses GFP in a subset of GABAergic neurons: DD, VD and RME. GFP expression
in these neurons was significantly higher in zag-1; unc-25::gfp than
in wild-type unc-25::gfp animals. Tryptophan hydroxylase catalyzes
the first step in serotonin biosynthesis and tph-1 is expressed in
serotoninergic neurons (Sze et al.,
2000). Although HSN lacked tph-1::gfp expression in
zag-1 mutants, expression in ADF, NSM, CP and R(1,3,9) was comparable
to wild type. However, unlike wild type, faint GFP expression was detected in
VC4 and VC5 in zag-1 mutants (35/100 VC4 and 57/100 VC5 zd85
zdIs13). VC4 and VC5 are detected weakly and sporadically with serotonin
antisera and express the vesicular monoamine transporter cat-1
(Duerr et al., 1999).
Therefore, in zag-1 mutants, GFP expression in VC4 and VC5 apparently
reflects upregulation of undetectable wild-type tph-1::gfp
expression, rather than inappropriate expression. Last, dat-1 encodes
a dopamine transporter homolog that presumably facilitates dopamine reuptake
(Nass et al., 2001). The
dat-1::gfp(zdIs31) strain had moderate GFP expression in the eight
dopaminergic neurons, ADE, PDE and CEP. Expression was greatly enhanced in
these neurons by zag-1 mutations. Together, these results indicate
that zag-1 activity downregulates the expression of neurotransmitter
biosynthetic and reuptake genes.

zag-1 molecular analysis

We cloned zag-1 by genetic mapping and transformation rescue of
its mutant phenotype (Fig.
3A,B). zag-1 maps between vab-2 and
dpy-13 on chromosome IV (see Materials and Methods). We
tested cosmids within this 469 kb interval by germline transformation and
identified a single cosmid, F28F9, that completely rescued the uncoordinated
phenotype of zag-1(zd85) animals. A 10 kb KpnI-SalI
subclone of F28F9, which encompasses the hypothetical gene F28F9.1, also
rescued. We analyzed the nucleotide sequences of the predicted coding regions
and splice junctions of F28F9.1 from wild-type and zag-1 mutants and
found that both zd85 and zd86 are G:C to A:T transitions
that generate termination codons in exon 5. Together, these data indicate that
zag-1 is F28F9.1.

We determined the DNA sequence of a full-length cDNA (provided by Y.
Kohara) to establish the zag-1-gene structure
(Fig. 3C). zag-1 has
seven exons, a five-nucleotide 5′ UTR and, at most, a 444-nucleotide
3′ UTR. Spliced leader 1 sequences in cDNA indicated that the
zag-1 transcript is trans-spliced. On the basis of the DNA
sequence, zag-1 encodes a 596-amino-acid protein with five C2H2-type
Zn-finger domains, two at the amino end and three at the carboxyl end, and a
single homeodomain located in the middle (hence zag-1,
Zn-fingerhomeodomain, axon guidance) (Fig.
3D, Fig. 4A). The N
and C-terminal Zn-finger-domain arrays are highly similar to each other, and
to the Zn-finger domains in Drosophila ZFH-1 and vertebrateδ
EF1 and SIP1 proteins (Fig.
4B). Although the total number of Zn-finger domains varies in
these proteins, the overall structure, namely, Zn-finger-domain clusters
positioned at the N and C terminals and a centrally located homeodomain, is
conserved and is a defining feature of the δEF1/ZFH-1 protein family.
The Zn-finger-domain clusters of δEF1/ZFH-1 proteins bind to the
consensus sequence CACCT, the E-box sequence CACCTG and tandem arrays of these
motifs (Ikeda and Kawakami,
1995; Remacle et al.,
1999; Sekido et al.,
1997), indicating that ZAG-1 can also probably bind to CACCT
sequences. The ZAG-1 homeodomain is most similar to those present in LIM
homeodomain proteins, such as C. elegans LIM-4 and MEC-3, and the
second homeodomain present in the C. elegans ZFH-2-related protein
encoded by ZK123.3 (Fig. 4C).
ZAG-1 lacks sequences characteristic of the Smad-interacting domain found in
vertebrate SIP1 orthologs (Verschueren et
al., 1999).

The zd85 and zd86 mutations generate termination codons
in exon 5 of ZAG-1. zd85 alters the glutamine codon for residue 380
(CAG → TAG) and zd86 changes the tryptophan codon for residue
369 (TGG → TAG). Both mutants are predicted to lack the C-terminal
Zn-finger domains but retain the N-terminal Zn-finger domains, the homeodomain
and the CtBP-interaction motif, and are likely to retain partial ZAG-1
function (see Discussion).

The C. elegans Sequencing Consortium has released preliminary
genomic sequence of the nematode C. briggsae
(www.wormbase.org),
allowing identification of a zag-1 ortholog. The C. elegans
and C. briggsae ZAG-1 proteins are similar (88% identity)
(Fig. 4A). The predicted amino
acid sequences of the Zn-finger domains, homeodomain and CtBP-interaction
motif are identical, whereas other regions contain conservative substitutions
and short deletions and insertions. The genomic organization of the two
zag-1 genes is similar and the positions of introns are identical
(Fig. 3C,
Fig. 4A). In addition to coding
region, sequence conservation is present upstream of the ATG and in several
introns.

Neurons and muscle express zag-1

We generated zag-1 GFP translational and transcriptional
transgenes to examine the expression pattern of zag-1
(Fig. 5A). We fused 9.3 kb of
zag-1 genomic sequence (which included 4 kb of upstream sequence) to
GFP coding and unc-54 3′UTR sequences to obtain a construct
that produced a full-length ZAG-1 protein tagged with GFP at its C-terminus
(Fig. 5A). This
zag-1::ZAG-1-GFP construct rescued zag-1 mutants (data not
shown), indicating that the fusion protein is functional and expressed in a
spatial and temporal pattern sufficient for rescue.

Examination of the zag-1::ZAG-1-GFP(zdIs39) strain revealed
relatively faint GFP expression in neuronal nuclei from mid to late
embryogenesis and during the L1 stage (Fig.
6A,B). We saw widespread expression in head and tail regions
containing differentiating neurons beginning around the comma stage that
diminished as embryogenesis continued. Expression remained in a few neurons at
hatching and was typically nonexistent by midlarval stages. We also detected
transient expression in the postembryonic Pn.a neuroblasts and their
descendants during the L1. We conclude that zag-1 is expressed
transiently in most or all neurons during embryogenesis and in the
Pn.a-derived ventral cord neurons during the L1, and that zag-1
expression coincides generally with the time period that neurons extend axons
and undergo terminal differentiation.

We constructed a translational fusion to exon 2 that expressed a GFP
chimeric protein containing the first 213 amino acids of ZAG-1.
zag-1::ZAG-1(213)-GFP (zdIs40) animals displayed a pattern of
neuronal expression similar to that of zag-1::ZAG-1-GFP(zdIs39)
animals, indicating that sequences between the end of exon 2 and 7 were
unnecessary for generating the observed expression pattern. The GFP signal was
detected in neuronal nuclei, showing that the first 213 amino acids of ZAG-1
are sufficient for nuclear translocation.

We examined the expression patterns of transcriptional GFP reporters
containing 0.3-4.0 kb of upstream zag-1 sequence using integrated or
multiple, independent extrachromosomal array-containing lines to investigate
further the regulation of zag-1 expression
(Fig. 5A). In contrast to the
expression patterns displayed by the translational reporters, using the 1.6
kb, 2.9 kb and 4.0 kb upstream fragments we observed bright, persistent, GFP
expression in a subset of neurons in the head and tail that began during
midembryogenesis and continued throughout larval development and adulthood
(Fig. 6C). These three
fragments produced largely similar neuronal expression patterns, although the
4 kb fragment directed expression in additional neurons in the head, including
the command interneurons. By contrast, the 0.3 kb and 0.7 kb fragments yielded
only weak expression in a few neurons in the nerve ring. These data imply that
sequences between -1.6 kb and -0.7 kb (where the first nucleotide of ATG is
+1) are required for most of the expression seen in the characteristic subset
of head and tail neurons.

We observed anal depressor and intestinal muscle expression starting around
hatching using either the 2.9 kb or 4.0 kb 5′ fragment. No expression
was seen using 1.6 kb or shorter fragments, indicating that sequences between
-2.9 kb and -1.6 kb are needed for expression in these enteric muscles. The
2.9 kb fragment produced transient expression in body-wall muscles, starting
before the comma stage and ending during midlarval development. The 0.3 kb,
0.7 kb, 1.6 kb and 4.0 kb fragments did not generate expression in body-wall
muscle, suggesting that sequences between -2.9 kb and -1.6 kb promote
body-wall-muscle expression and that sequences between -4.0 kb and -2.9 kb
repress this.

The 4.0 kb zag-1::gfp transcriptional and
zag-1::ZAG-1(213)-GFP translational reporters share the same 4 kb
upstream sequences but differ because zag-1::gfp includes the coding
sequences for the first seven amino acids of ZAG-1 (as do all the
transcriptional reporter constructs) and zag-1::ZAG-1(213)-GFP
includes sequences for exon 1, intron 1 and most of exon 2. Thus, exon 1
through exon 2 sequences are needed to confer the transient, widespread, weak
GFP expression that is characteristic of the two translational reporters.
Although there are differences in the structure and localization of the fusion
proteins, we believe that transcriptional mechanisms mediated by sequence
elements in intron 1 are most likely to underlie the reduced, transient
expression, as detailed below. Together, these results indicate that
zag-1 is expressed broadly in the nervous system and in selected
muscles. The different expression patterns of the translational and
transcriptional constructs reveal the presence of multiple regulatory elements
in the promoter and in the gene that either upregulate or downregulate
zag-1 expression.

zag-1 mutants lack muscle defects

Expression of zag-1 in neurons and muscle is reminiscent of the
neural and mesodermal expression of Drosophila zfh-1 and vertebrateδ
EF1. zfh-1 mutations perturb development of gonadal mesoderm,
heart and other mesodermally derived tissues, and δEF1 is a negative
regulator of muscle differentiation in vitro. We examined the morphology and
development of body-wall muscle using the talin::gfp(zdIs15)
reporter, which produces high GFP levels in all body-wall muscles starting
during embryogenesis. GFP expression and muscle morphology were similar in
wild-type talin::gfp and zag-1; talin::gfp animals.
TGFβ UNC-129 is expressed in dorsal but not ventral body-wall muscles
(Colavita et al., 1998); the
body-wall expression pattern of unc-129::gfp(zdIs42) was unaffected
by zag-1 mutations. The morphology of anal depressor and intestinal
muscles was unchanged in zag-1 mutants when observed using a
zag-1::gfp reporter. Thus, although zag-1 is expressed in
anal depressor, intestinal and body-wall muscles, we failed to detect defects
in either muscle development or differentiation in zag-1 mutants
using these reporters.

ZAG-1 represses zag-1 expression

We examined zag-1 GFP reporter expression in zag-1
mutants to investigate whether ZAG-1 regulated its own expression. We found
that zag-1 mutations affected neuronal expression patterns of the 1.6
kb, 2.9 kb and 4.0 kb upstream fragments but not the 0.3 kb and 0.7 kb
fragments (Fig. 5A). In
particular, we found that zag-1 mutations induced bright GFP
expression in the Pn.a-derived VNC motor neurons that began during midL1 and
continued throughout adulthood (Fig.
6E). We also observed at least seven neurons in the tail that
expressed GFP compared to only PVQ and PVT in wild type, as well as many
additional GFP-expressing neurons in the head, including the command
interneurons. Therefore, zag-1 activity represses zag-1::gfp
expression in many neurons via sequences located between -1.6 kb and -0.7 kb.
The finding that the 4.0 kb upstream fragment also directed expression in the
command interneurons in wild-type animals indicates the presence of multiple,
positive and negative regulatory elements for command interneuron expression.
zag-1 mutations did not alter zag-1::gfp expression in anal
depressor, intestinal and body-wall muscles. The expression pattern of the
zag-1::ZAG-1(213)-GFP reporter, which did not rescue zag-1,
was also unaffected by zag-1 mutations. The inability of
zag-1 mutations to alter zag-1::ZAG-1(213)-GFP expression
indicates that either the repression mediated by sequences in exon 1 through
exon 2 is independent of zag-1 activity or our two zag-1
mutants retain partial activity.

δEF1/ZFH-1 proteins bind to the consensus sequence CACCT, the E box
sequence CACCTG and tandem arrays of these motifs
(Ikeda and Kawakami, 1995;
Remacle et al., 1999;
Sekido et al., 1997).
Examination of zag-1 genomic sequences from C. elegans and
C. briggsae revealed several blocks of identity in addition to
conserved sequences in coding regions. Several, conserved CACCT motifs are
present in the 0.9 kb region that is essential for ZAG-1-mediated repression
of zag-1::gfp expression and in the first, third and fourth introns
(Fig. 3C,
Fig. 5B). Because previous
studies have shown that altering the CACCT sequence to CATCT either greatly
reduced or eliminated binding of either N or C-terminal δEF1/ZFH-1
Zn-finger-domain clusters (Remacle et al.,
1999), we introduced these mutations into the 1.6 kb promoter
fragment to test whether these sites influenced zag-1 expression. We
altered the conserved E box (CACCTG) at -1114 and the two CACCT sites at -918
and -895, either singly or together, and examined GFP-reporter expression.
Alteration of the E-box site had no obvious effect on expression, whereas
mutation of either CACCT motif had a moderate effect. Both -918A and -895T
promoter mutants produced reproducible expression in the command interneurons
and, occasionally, generated expression in one or two ventral cord motor
neurons in animals that contained extrachromosomal arrays. In the CACCT double
mutant (-918A, -895T), the pattern of expression was strikingly similar to
that observed in zag-1 zag-1::gfp mutants, including expression in
command interneurons and most or all ventral cord motor neurons. The 4.0 kb
fragment containing the -918A and -895T CACCT mutations also produced an
expression pattern comparable to that observed in a zag-1-mutant
background (Fig. 6D-F). Based
on these results and published studies of the binding of δEF1/ZFH-1
proteins, we conclude that the tandem CACCT site is a ZAG-1 binding site and
that ZAG-1 directly represses expression via this site.

DISCUSSION

zag-1 activity establishes several neuronal characteristics, such
as cell position, axonal structure and gene-expression profile. Although
zag-1 mutations confer various defects on sensory, motor and
interneurons, common or related phenotypes are evident. These include the
absence of stereotypic axon branches and upregulation of neurotransmitter
biosynthetic and reuptake genes. zag-1 functions less to define
neuron identity per se and more to generate features characteristic of a
particular type of neuron. The specificity and selectivity of zag-1
phenotypes for each neuron type suggests that zag-1 acts in
combination with other cell-type-specific factors to control
differentiation.

SRA-6 is a candidate chemosensory receptor, based on its predicted seven
transmembrane domain topology and expression in amphid sensory neurons ASH and
ASI (Troemel et al., 1995).
sra-6::gfp provides an ideal indicator of PVQ development and
differentiation, although sra-6 function in interneuron PVQ is
unclear. zag-1 is required for specific elements of PVQ
differentiation: LIM homeodomain gene lin-11 expression and axonal
development appear wild type, whereas sra-6 expression is either
reduced or eliminated. Because there is no evidence that ZAG-1 acts as a
transcriptional activator, regulation of sra-6 expression is,
presumably, indirect. PVQ sra-6 expression also requires PAG-3, a
Zn-finger-domain protein that functions in neural differentiation and cell
lineage (Cameron et al., 2002;
Jia et al., 1997). Although
zag-1 and pag-3 are coexpressed in other neurons, they do
not share other known phenotypes.

zag-1 mutations disrupt several late differentiation features of
HSN, including axon pathfinding, hood formation and tph-1 expression.
Early development of HSN appears unaffected because HSNs migrate to their
correct, midbody position, express unc-86 and do not misexpress
odr-2. zag-1 HSN phenotypes are remarkably similar to those of
egl-45, sem-4 and unc-86
(Desai et al., 1988). The
Zn-finger-domain gene sem-4 controls neuronal and mesodermal
development (Basson and Horvitz,
1996) and the POU-homeodomain gene unc-86 regulates
neuronal-cell lineage and differentiation
(Finney et al., 1988).
egl-45, sem-4 and unc-86 act in a genetic pathway containing
both parallel and overlapping branches that controls HSN development
(Desai et al., 1988).
zag-1 likely functions downstream of or in parallel to
unc-86 because zag-1 mutations do not affect
unc-86::gfp expression.

Ectopic HSN odr-2 expression in zag-1 adults reveals that
zag-1 activity blocks expression of nonHSN genes as well as promotes
HSN differentiation and proper gene expression. Regulation of odr-2,
which encodes a GPI-linked cell-surface protein, is neuron-type specific; that
is, zag-1 mutations induce misexpression of odr-2 in HSN
and, occasionally, PVM but do not otherwise appear to alter the odr-2
expression pattern. Similarly, zag-1 is needed for proper expression
of the glutamate receptors GLR-1 and NMR-1 and the ion channel MEC-4, and to
prevent misexpression of GLR-1 and NMR-1.

zag-1 activity downregulates the expression of neurotransmitter
biosynthetic and reuptake genes. Although zag-1 does not determine
which neurons express dat-1, tph-1 and unc-25, it modulates
expression levels in the appropriate neurons, acting, perhaps, as a `gain'
switch. zag-1 expression appears to be restricted to embryos and L1s,
but upregulation of expression is still apparent in adults, which indicates
that either ZAG-1 is present but undetectable in adults or ZAG-1-established
expression levels are maintained.

zag-1 is essential for the formation of stereotypic axon branches
of several neuron types. zag-1 mutations block the generation of
anteriorly directed branches of ADE, ALM and AVM and the posteriorly directed
branch of PDE. Except for lacking these branches, the axon trajectories of
these neurons appear wild type and full-length, indicating an explicit defect
in the development of axon branches rather than a deficiency of axon
outgrowth. Although HSN, DD and VD axon-branching defects are coupled with
broader pathfinding errors, we believe that these defects reflect a specific
role of zag-1 in branching and pathfinding. The dorso-ventrally
oriented ADL branches were unaffected by zag-1 mutations (data not
shown), indicating that zag-1 is not required for all axon-branching
patterns.

The dramatic guidance, branching and fasciculation defects of many
glr-1::gfp-expressing interneuron and motor neuron axons illustrate
further the role of zag-1 in creating axonal structures. Exuberant
extension and branching might reflect either a guidance defect or an inability
to terminate outgrowth at appropriate targets. The defasciculation of the
nerve ring and ventral nerve cord and premature termination of the AVG axon
reveal that zag-1 is also required for nerve bundle formation and
axon outgrowth.

The mature axonal morphology of C. elegans neurons has been
described in great detail, although the assembly of these axonal structures is
less well understood. For example, several nonexclusive ways to sculpt the
mature ALM axon structure are possible: ALM growth cone might first extend to
the first bulb of pharynx and a collateral branch might later enter neuropil;
ALM growth cone might first project to nerve ring, turn and enter neuropil and
a collateral branch might later project anteriorly to first bulb of pharynx;
and ALM growth cone might extend to nerve ring, split and form both branches
concurrently. The latter two scenarios are consistent with the observation
that zag-1 mutants lack anterior but not neuropil branches. We
analyzed axonal structures in young adult hermaphrodites and limited analysis
of larvae revealed similar axonal defects. Our current results indicate that
zag-1 mutations block the formation of axon branches but do not rule
out the possibility of inappropriate axon-branch retraction or pruning.
Real-time analysis is needed to resolve the axon assembly pathway of ALM and
other neurons, but this is technically difficult at present. However,
regardless of assembly pathway, zag-1 activity is needed to shape the
mature axonal configuration.

HSN has two projections during the L2 and L3, one directed ventrally that
will ultimately reach nerve ring and be maintained in adult, and one directed
dorsally that is, presumably, retracted later because it is not found in
adults (Garriga et al., 1993).
The HSNs often have two processes in zag-1 mutants, supporting the
notion that zag-1 activity sculpts the mature axonal morphology by
promoting the formation of new branches, preventing extension of inappropriate
processes and eliminating immature structures.

Interactions with vulval cells control HSN-axon branching
(Garriga et al., 1993) and
interactions with BDU guide AVM branch into the mature neuropil
(Walthall and Chalfie, 1988).
Similar cellular interactions are believed to guide the formation of other
axon branches. zag-1 might act in either a signaling or responding
neuron to coordinate branch formation and pathfinding interactions. The
specificity of axon-branch defects in zag-1 mutants indicates the
existence of a branch-formation program that functions in combination with a
primary axon-guidance program to define a particular axonal-projection
pattern. Such branching programs might function either during or subsequent to
formation of primary axons and entail the regulation of genes that generate,
recognize and transduce spatial and temporal branching signals. The branching
defects of ADE, PDE, ALM and AVM might represent a failure to activate a
branching program, whereas defects of HSN, VD and
glr-1::gfp-expressing neurons might reflect coexpression or
misexpression of primary axon-guidance and branching programs.

ZAG-1 is a transcriptional repressor

ZAG-1 is a δEF1/ZFH-1 Zn-finger-homeodomain protein. Mutation of
either zag-1 or two, conserved CACCT sites in the zag-1
promoter upregulates zag-1::gfp expression in many neurons, including
ventral cord motor neurons and command interneurons. As Zn-finger-domain
arrays from δEF1/ZFH-1 proteins bind CACCT sequences, we conclude that
ZAG-1 represses zag-1 expression by binding directly to this tandem
CACCT site. Although many direct target genes of δEF1/ZFH-1 and SIP1
have been identified (reviewed by van
Grunsven et al., 2001), our results provide the first example thatδ
EF1/ZFH-1 proteins regulate their own expression. In addition, our
findings that both CACCT sequences are needed for complete repression are
consistent with studies of Remacle et al.
(Remacle et al., 1999), which
show that SIP1 binds as a monomer and contacts one CACCT site with one
Zn-finger-domain cluster and the other CACCT(G) site with the second
cluster.

δEF1-knockout mice have defects in skeletal and thymus development,
whereas a δEF1δC727 mutant, which expresses
a δEF1 protein lacking the C-terminal Zn-finger-domain cluster like the
two ZAG-1 mutant proteins, has defects in only thymus development
(Higashi et al., 1997;
Takagi et al., 1998). These
results suggest that δEF1δC727 retains
sufficient activity to promote skeletal development and that δEF1/ZFH-1
binding sites can be divided into two classes: those that require the
C-terminal Zn-finger-domain cluster and those that do not. We infer that our
two ZAG-1 mutants also retain activity and regulate the subset of
zag-1 target genes that possess the second class of ZAG-1 binding
site. Conserved CACCT(G) motifs in the first, third and fourth introns
represent candidate ZAG-1-binding sites; however, zag-1 mutations
fail to alter expression of zag-1-ZAG-1(213)-GFP, which contains
intron 1 sequences. These observations indicate that these sequences are not
ZAG-1-binding sites and that other factors mediate repression or that these
sequences belong to the second class of ZAG-1-binding site. Our conclusion
that the truncated ZAG-1 proteins retain activity is consistent with the
observation that a zag-1 deletion mutant is not viable because of a
feeding defect (Wacker et al.,
2003).

Our GFP-transgene studies indicate that zag-1 is expressed
transiently in most or all neurons during embryogenesis and in the
Pn.a-derived ventral cord neurons during the L1. In general, the time of
zag-1 expression coincides with the period that neurons extend axons
and undergo terminal differentiation. The analysis of the
zag-1-promoter region indicates that zag-1 is also expressed
in anal depressor, intestinal and body-wall muscles, although no obvious
muscle defects were observed. Similarly, although Drosophila zfh-1 is
expressed in motorneurons, defects in motorneuron development have not been
identified in zfh-1 mutants. It is speculated that the lack of
neuronal and muscle defects in δEF1-knockout mice reflects a redundancy
with SIP1, which is also expressed in these tissues
(Postigo and Dean, 2000).
Examination of zag-1-null mutants and additional
muscle-differentiation markers is needed to investigate further the role of
zag-1 in muscle development

ZAG-1 and other neuronal differentiation regulators

Numerous transcriptional regulators have been identified that control the
generation, specification and differentiation of the 302 neurons of C.
elegans. Mutation of these factors cause a variety of defects that affect
different neuron types as well as different aspects and phases of neuronal
development. Although ZAG-1 shares some activities and characteristics with
known factors, the overall role of ZAG-1 in neuronal development is unique.
For example, zag-1 mutants have HSN-differentiation defects that are
similar to unc-86 mutants and PVQ-differentiation defects that are
similar to pag-3 mutants. However, unlike zag-1 mutations,
unc-86 and pag-3 mutations also cause some daughter cells to
reiterate the lineage of their mother, demonstrating that UNC-86 and PAG-3
control the generation of particular neuron types. Touch-receptor neurons
exhibit none of their unique differentiated features in LIM-homeodomain gene
mec-3 mutants (Way and Chalfie,
1988) but display only a subset of defects, such as errors in
mec-4 expression, cell migration and axon-branch formation, in
zag-1 mutants. Thus, ZAG-1 is not involved in generating neurons or
in defining every trait of a particular neuron type. In contrast to
lim-4 and unc-130 mutations, which cause specific neurons to
adopt alternative or default cell fates
(Sagasti et al., 1999;
Sarafi-Reinach and Sengupta,
2000), zag-1 mutations do not induce cell-fate
transformations but, rather, prevent the acquisition of a subset of
neuron-type characteristics. The function of zag-1 is most similar to
lim-6, unc-30 and unc-42, which establish select aspects of
late differentiation such as axonal development, synaptic connectivity and
neuron-type-specific gene expression (Baran
et al., 1999; Brockie et al.,
2001; Hobert et al.,
1999; Jin et al.,
1994). However, LIM-6, UNC-30 and UNC-42 have more restricted
patterns of expression compared to ZAG-1, which appears to be expressed in
most or all neurons. Thus, ZAG-1 acts as a global regulator of neuronal
differentiation and is the first transcription factor to be identified that
controls axon-branch formation.

Although most of the identified regulators are likely to function as
transcriptional activators, ZAG-1 acts as a transcriptional repressor. Other
factors involved in late-neural differentiation that repress transcription
directly include the homeodomain UNC-4 and Groucho-like corepressor UNC-37,
which specify synaptic choice (Winnier et
al., 1999). ZAG-1 might function as a temporal switch during
neuronal development; that is, ZAG-1 might initiate late differentiation by
turning off a `late-differentiation repressor' and/or genes involved in early
differentiation. The ectopic expression of glr-1, nmr-1 and
odr-2 in zag-1 mutants indicates that ZAG-1 also blocks the
adoption of inappropriate neuronal characteristics. As with specification of
neuron fate, combined actions of both transcriptional activators and
repressors are needed to establish the characteristics of terminal
differentiation. Last, the observation that a presumptive ZAG-1-binding site
is conserved between C. elegans and C. briggsae indicates
that a bioinformatics strategy can be used to identify potential direct gene
targets of ZAG-1, which will provide further insight into zag-1
function.

Acknowledgments

We thank Katrina Sabater and Ray Squires for generating plasmid constructs
and integrated transgenic strains, and B. Prasad, P. Sengupta and members of
the Clark laboratory for comments on manuscript. We are grateful to C.
Bargmann, A. Chisholm, J. Chou, A. Coulson, M. Driscoll, A. Fire, Z. Gitai, M.
Han, O. Hobert, Y. Jin, Y. Kohara, V. Maricq, T. Sarafi-Reinach and P.
Sengupta for providing strains and/or clones. We are eternally indebted to the
C. elegans Sequencing Consortium (Sanger Institute and Genome
Sequencing Center, Washington University, St Louis) for providing C.
elegans and C. briggsae genomic sequence. Some nematode strains
used in this work were provided by the Caenorhabditis Genetics Center. This
work was supported by grants from the Alfred P. Sloan Foundation, the March of
Dimes, the New York City Council Speaker's Fund and the NIH/NINDS (R01
NS39397).

Yochem, J., Gu, T. and Han, M. (1998). A new
marker for mosaic analysis in Caenorhabditis elegans indicates a
fusion between hyp6 and hyp7, two major components of the hypodermis.
Genetics149,1323
-1334.

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We are currently seeking proposals for four Workshops to be held in 2020. Do you have an idea for a Workshop? Please let us know and you could be one of our 2020 Workshop organisers. You focus on the science, we focus on the logistics. We are particularly keen to receive proposals from postdocs. Deadline date for applications is 25 May 2018.

Development is a proud sponsor of the upcoming Santa Cruz Developmental Biology Meeting, which takes place 11-15 August 2018 at the University of California, Santa Cruz . Registration for this meeting is now open!

Meet the preLighters! In the latest interview with our preLights community, the preLights team caught up with James Gagnon, Assistant Professor at the University of Utah, to talk about his research, how science can be made more open, his enthusiasm for the preLights project and the fun sides of being a junior PI.

To investigate which signalling pathways are regulated by nitric oxide during mouth development in Branchiostoma lanceolatum (amphioxus), Filomena Caccavale used a Travelling Fellowship from Development to visit The Oceanographic Observatory in Banyuls-sur-Mer, France, an area with a thriving natural amphioxus population. Read more on her story here.

Where could your research take you? Join Filomena and apply for the next round of Travelling Fellowships from Development by 25 May 2018.